Human and animal diseases can often be treated by supplying drug substances that correct disease symptoms by supplying a biochemical substance not made properly by the body. Examples include but are not limited to enzyme replacement therapies (ERTs) for the treatment of Lysosomal Diseases (LDs) like Hurlers Syndrome (MPS I) or Alzheimer's with corrective enzymes for these genetic or age related developmental diseases. These types of diseases are associated with failure of metabolic processes or function of specific cells and biochemical pathways within the body. A goal of many drug advancement programs is, therefore, focused on directing drugs more efficiently to the specific cells or tissues that are responsible for these functions. And the discovery described in this patent application is directed toward facilitating this goal.
Many plants produce specific carbohydrate binding lectins, agglutinins, and toxalbumins which are “AB” toxins that comprise a toxic A subunit protein (e.g., a ribosome-inactivating protein) and a non-toxic B subunit that is typically a lectin (carbohydrate binding protein) responsible for binding to the target cell surface, triggering endocytosis, and mediating intracellular trafficking. A classic example of this AB class of toxins is ricin toxin (from Castor beans) which incorporates a ricin A subunit (RTA) having ribosome-inactivating activity and a ricin B subunit (RTB) having galactose- and galactosamine-binding activity that directs cell uptake and trafficking. As with other AB toxins, the lectin binding capacity of cell surfaces allows binding with carbohydrate binding domains of lectin sequences on the RTB subunit of Ricin triggering endocytosis of the toxin into the cell and transcytosis of the toxin across cell layers by several different mechanisms including Receptor-Mediated Transcytosis (RMT) and Adsorptive-Mediated Transcytosis (AMT).
The development of effective therapeutic drugs to treat many diseases that produce significant impairment of cellular and metabolic function would be facilitated by the discovery of new ways to deliver these drugs to sites of disease pathology in cells and organs of the body. Lysosomal Diseases (LDs), also commonly called lysosomal storage diseases (LSDs) are representative of this class of diseases. LDs are a group of approximately 50 rare inherited metabolic disorders that result from defects in lysosomal function (Winchester et al. (2000)). Lysosomal storage diseases result when a specific organelle in the body's cells—the lysosome—malfunctions. Lysosomal storage disorders are caused by lysosomal dysfunction often as a consequence of deficiency of a single enzyme required for the metabolism of both large molecules or small compounds such as lipids, glycoproteins (sugar containing proteins) or so-called mucopolysaccharides. The lysosome is commonly referred to as the cell's recycling center because it reprocesses or catabolyzes metabolic waste material into substances that the body can recycle into useful substances or eliminate though the kidneys and urinary tract. Lysosomes break down this waste matter via enzymes that are highly specialized proteins that perform most of the chemical reactions essential for cellular functions. Lysosomal disease disorders are triggered when a particular enzyme is defective and suffers from loss of function or is present in too small amounts to perform normal metabolic functions or is missing altogether. When this happens, waste substances may build up to toxic levels that interfere with normal healthy metabolism leading to serious disease symptoms and even death.
Defects in cellular metabolic machinery caused by genetic mutations of lysosomal enzyme genes are a major cause of LDs and are usually expressed in all cells of the body. The level of threat to life of genetically defective lysosomal enzymes varies, however, depending on how much the mutation reduces the function of a particular enzyme and what bodily functions are most impaired. Research during the last few decades has led to the development of effective enzyme replacement (ERT) therapeutics drugs whereby defective enzymes in LD patients are replaced by intravenous delivery of normal enzymes produced by recombinant manufacturing technologies. Although these drugs are now available for a half a dozen of LDs their effectiveness in reducing disease symptoms varies depending on which cells and organs are affected, the severity of symptoms, and the stage of disease progression. Current drugs are more effective in certain organs such as liver and spleen but often much less effective in treating symptoms in such organs as bone, heart, lungs, kidneys, and the CNS (central nervous system) including brain where severe symptoms occur in many LDs. The invention described in this patent is designed to more effectively target ERT's to these recalcitrant organs and also improve treatments in organs such as liver where current therapies could use improvement.
Poor brain development and neural degeneration of the brain and CNS are some of the most devastating symptoms in LDs. The effect of LDs on brain function has been well studied and is a representative example of currently untreatable symptoms of LDs. Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function. It is maintained by the blood-brain barrier (BBB) which regulates the transport of molecules from blood into brain and backwards. The function of this highly specialized barrier is to (1) protect the brain from blood-borne substances that are potentially detrimental to brain function and (2) to provide nutrients and other required substances to the brain parenchyme by specialized transport systems. The main structures responsible for this barrier property are the tight junctions (TJ). TJ are highly developed in endothelial cells of the brain and CNS vasculature but only moderately formed between endothelial cells of the peripheral vasculature: leaky blood capillaries in the body allow many molecules to cross through to tissue, but the TJ construction of the vessels in the CNS guards against this less restricted entry to the brain (Förster (2008)).
The tight control in transport of chemicals and proteins across the BBB poses a significant challenge to the delivery of diagnostic/therapeutic proteins, nucleic acids, and other drugs to the brain. Small molecules such as lipophilic drugs, gases, glucose, and essential nutrients cross the BBB by a number of passive and active transport mechanisms. In contrast, macromolecules such as proteins and nucleic acids are generally excluded from the brain and only a selective subset of proteins is transported across the BBB using either Receptor-Mediated Transcytosis (RMT) or Absorptive-Mediated Transcytosis (AMT). For substances transported via RMT mechanisms, a specific receptor (e.g., the insulin receptor or the transferrin receptor) is present on the luminal surface of the CNS endothelial cells which mediates uptake, transcytosis, and release of proteins or other therapeutic substances at the abluminal or basal surface where they can access the glial and neuronal cells of the brain. RMT mechanisms are “saturable” and the amount of product and rate by which substances can be mobilized across the BBB are limited by the number of available receptors present on the luminal surface.
In contrast to RMT mechanisms, Absorptive-Mediated Transcytosis (AMT) is independent of specific receptors and involves the binding of specific proteins or substances to the endothelial cell surface by interactions that trigger endocytosis and vesicular trafficking such that a proportion of the endocytosed substance is carried across the endothelial cell layer and subsequently released on the basal/abluminal side providing access to cells of the CNS. The selectivity and control of AMT mechanisms are not well understood but proteins such as cationated albumin and the TAT protein of HIV are known to enter the brain by this mechanism. AMT is considered non-saturable and may have the potential to deliver 10-fold greater amounts of product across the BBB compared to transport via the RMT. The present invention has the advantage that it can utilize multiple trans-cellular transport mechanisms including the AMT and RMT systems.
Role of Lectin in Toxicity of the AB Toxins Such as Ricin:
Many plant derived AB toxins are toxic because they inhibit protein synthesis and ricin toxin is considered a model of this class which includes, but is not limited to, ricins, abrins, nigrins, the mistletoe lectins and the viscumin toxins, ebulins, pulchellin, pharatoxin, hurin, and phasin toxins. Many, but not all, of these protein toxins are dimers made up of A and B protein subunits. Subunit A is the actual toxin, while subunit B is a lectin (carbohydrate-binding protein) that helps deliver the toxic subunit protein inside cells by binding to components on the cell surface or cell membrane and triggering uptake by cells. Once inside a cell, the subunit A protein of ribosome inactivating toxins like Ricin is able to selectively catalyze the cleavage of an N-glycosidic bond in the 28S ribosomal RNA that is a crucial part of eukaryotic ribosomes (en.wikipedia.org/wiki/ribosome), the organelles inside cells that make proteins, thus inhibiting protein synthesis and essentially shutting down the cell.
AB toxins may enter the body through many routes including via mucosal surfaces such as the gut, nose, lungs or may be administered transdermally or by injection. Research on the metabolism of AB toxins in animals has led to key insights in the uptake of proteins and other compounds into animal cells. For example, ricin toxin targets cells with galactose residues on their external surfaces. Research studies have identified at least five different biochemical uptake mechanisms. These studies have shown that ricin uses both dynamin-dependent and -independent routes of uptake into cells. Additionally, ricin has been observed to trigger endocytosis by interaction with the high mannose receptors based on its own mannose terminated glycans in addition to clathrin-dependent and -independent pathways. Clearly an important feature leading to the effectiveness of AB plant toxins in animals is the specialization of the A and B subunits of these proteins and the functional optimization of each subunit (A subunit: toxicity and B subunit: delivery) presumable thru evolution. Because of toxicity of AB toxins they have not been exploited systematically in drug discovery programs; this patent presents an invention which overcomes this drawback.
There are also other classes of lectins that do not specifically comprise AB toxins but possess lectin-mediated ability to bind to cell surface components and to direct uptake into cells and transcytosis across cells and to carry or deliver associated molecules. The best characterized lectins in this class typically have been identified from plants and include, but are not limited to, lectins such as wheat germ agglutinin, phytohemagglutin, Concanavalin A, the peanut and soybean lectins, and Jacalins.